Molecular imprinting of synthetic polymers is a process where functional and cross-linking agents (typically monomers) capable of polymerizing are copolymerized in the presence of a target molecule, which acts as a molecular template. Before polymerization, the functional monomers either form a complex with the template via non-covalent interactions, or are covalently coupled forming a polymerizable derivative of the template. After polymerization, the functional groups of the monomers are held in position by the highly cross-linked polymeric structure. Subsequent removal of the template by solvent extraction and/or chemical cleavage reveals binding sites that are complementary in size and shape to the target molecule. In this way, a molecular memory is introduced in the polymer (now termed a “molecular imprinted polymer” or “MIP”), which is now capable of rebinding the target with very high specificity.
Originally, MIPs were employed as stationary phases in HPLC, notably for chiral separation. Subsequently, their use has been extended to other analytical techniques such as thin layer chromatography, capillary electrophoresis, solid-phase extraction, and immunoassay type binding assays. The binding sites often have affinities and selectivities approaching those of antibody-antigen systems. These mimics display some clear advantages over antibodies for sensor technology. Because of their highly cross-linked nature, MIPs are intrinsically stable and robust, facilitating their application in extreme environments, such as in the presence of acids, bases, or metal ions, in organic solvents, or at high temperatures and pressures. Moreover, MIPs are relatively inexpensive to produce and can be stored in a dry state at room temperature for long periods of time.
Hence, in principle all MIPs are made in the following way: Monomers (or polymerizable agents) and target (or template) molecules are mixed, self assembly occurs, cross binder is added and polymerization can be initiated. After polymerization the polymer is typically (but not always, cf. below) broken down into small fractions and the target molecule is extracted. If the MIPs are put into a solution of target molecules these will rebind to the MIPs (cf. also: Yu Cong; Leif Schweitz; and Ioana Wärnmark-Surugiu).
History of MIPs
One of the first examples of MIP preparation was described as early as in 1949 (Dickey) who used a kind of silica (water glass) for selective recognition of dyes. Much later other kinds of self-organizing systems to build up networks wherein it was possible to bind targets/analytes specifically were described (Ramström et al., Schweitz et al., and Vlatakis et al.)
Choice of Monomers and Polymerization
In the 1970s and 1980s (cf. Shea 1986, Shea 1990 and Wulff 1987) the concept of covalently binding the template/target molecule directly to the polymer used for building the scaffold was described. The claim was that the direct binding would lead to a more homogeneous distribution of binding sites throughout the polymer. However, at the same time this leaves the problem of removing the template after the polymerization. In order to remove the template both a micronization of the polymer and a chemical bond breaking is needed.
Preparing MIPs without tethering the template to one of the monomers used during polymerization often results in good MIPs but the experience in literature is that a lot of the MIPs particles will contain binding sites that tend to bind the template/analyte in less specific parts of the molecule and hence not giving the desired specificity of the resulting MIPs. This is very important for MIPs used for analytical purposes especially if the object is to separate stereoisomeric forms of molecules, whereas this is less important in the case where the main objective is to enhance the total binding capacity of the resulting MIPs.
In certain analytical situations it has been proven that the template has to be of a different identity i.e. instead of using the actual template the produced MIPs are build over template “mimics” in order not to pollute the sample to be analyzed. It is clear that identification of a template mimic that is capable of ensuring a specific binding between the analyte and MIP can constitute a difficult task.
A completely different method of preparing MIPs is by polymerizing the mixture while the monomer, cross binders and template (or template mimics) are kept in particulate format in an emulsion hence leaving the resultant MIP as a particle directly (Funke et al.). The particle size of MIP made with this process will depend on, amongst other things, the monomer concentration and the stirring rate (determining the droplet size in the emulsion). (In order to get particle sizes down to 1 μm one needs to stir the solution at more than 1000 rpm). According to literature the disadvantages with this type of processes are long preparation times and low yields.
In general, the prior art often describes the difficulties of preparing reproducible MIPs where both the capacity and the specificity is not compromised (hence lower than desired).
U.S. Pat. No. 4,111,863 describes “A non-swellable three-dimensional polymer having a component which is a residue of an optically active compound, which residue is chemically removable from said polymer to leave behind in the physical structure of said polymer a void corresponding to the size and shape of said residue of optically active compound, and a particular steric arrangement of functional groups within the void of said polymer corresponding to the chemical structure of said residue of optically active compound . . . .” The “optically active compound” being the template that the MIP intentionally should be able to bind subsequently.
In U.S. Pat. No. 5,110,833 “A method of producing synthetic enzymes or synthetic antibodies, comprising the orientation of monomers around a print molecule, addition of crosslinkers, polymerizing to a polymer and subsequent removal of the print molecule, thereby creating a cavity in the polymer corresponding to said print molecule” is claimed to increase specificity of the MIP towards the template molecule. In other words, the performance improvement claimed in U.S. Pat. No. 5,110,833 is based on optimizing the contact between the template molecule and monomer units prior to polymerization.
In U.S. Pat. No. 6,881,804, introduction of porosity in the MIP is described as a means to increase to performance of a MIP by increasing the access to the void that is intended to interact with the template.
In U.S. Pat. No. 6,638,498 specifically selected monomers are claimed for generation of bile acid specific MIP's and in US 2004/0157209 A1, it is suggested to immobilize the template molecule on a support material prior to polymerization. All of the suggestions to improve the performance of MIPs deal with the chemical characteristics of the monomers or the architecture of the MIPs, which are all process steps that take place prior or during the preparation of the MIP.
U.S. Pat. No. 5,994,110 discloses MIPs, which are produced in situ to form small polymers/oligomers, which include a structure complementary to a template molecule. The polymers or oligomers form a coating or image around the biomolecule, which coating or image is removed therefrom, and discrete entities are derived therefrom, which may be used, e.g., as therapeutic or prophylactic agents, i.e. drugs. Due to this type of production process, U.S. Pat. No. 5,994,110 does not utilise a micronization step as in conventional MIP particle preparation. U.S. Pat. No. 5,994,110 does suggest separation of MIPs from non-binders, but the methods suggested all rely on the very small size of the MIPs produced e.g., via chromatography but only when the MIPs are soluble entities. It is e.g. specifically indicated that therapeutically active MIPs according to U.S. Pat. No. 5,994,110 are those which exhibit molecular weights in the lower end of the 1-200 kDa range. Further, U.S. Pat. No. 5,994,110 does not disclose any means for separating suspended insoluble MIPs into “good binders” on the one hand and “less effective or non-binders” on the other.
US 2009/0194481 relates to a composite material obtainable by agglomerating previously prepared MIPs.
The present assignee has previously filed WO 2007/095949, which relates to preparation of MIP compositions having improved affinity for a template/target. In brief, the method entails subjecting insoluble MIPs prepared by traditional means including micronization as described above and then subsequently subjecting these to an affinity purification procedure, which is adapted to affinity purify insoluble material; useful technologies discussed in WO 2007/095949 are expanded bed adsorption and agglutination. This technology provides for MIP compositions where all or substantially all MIPs in the compositions bind the same target agent, since it was found that prior art insoluble MIP compositions includes large fractions of MIPs which binds only weakly or not at all to the intended target agent.
The present assignee has also previously filed WO 2011/033021, which relates to a purification scheme useful for multi-specific receptors such as MIPs—the method utilises at least 2 successive rounds of affinity purification where the capture agent in the affinity purification is coupled via different functionalities to a support in each of the at least two rounds. This technology ensures that only mulitispecific receptors that bind all relevant binding sites on the capture agent pass the entire purification process as the binding sites which may be “hidden” from interaction with the receptors during the first round of purification are exposed for binding to the receptors in the subsequent round(s). The technology in WO 2011/033021 has in particular been devised in order to allow preparation of MIPs that target amino acids such as phenylalanine.